Abstract: Our goal is to introduce a new class of genetically encoded optical indicators, based on the huge diversity of environmentally sensitive spectral shifts that occur naturally in microbial rhodopsin proteins. We recently created a microbial rhodopsin-based fluorescent indicator of pH, with a sensitive range from pH 6.8 to 8.8. We have preliminary results on a microbial rhodopsin-based indicator of membrane potential, which shows greater sensitivity than any existing optical sensor of membrane potential. Just as GFP revolutionized biology through its ability to track the positions of proteins in cells, we believe that microbial rhodopsins will have a broad impact through their ability to transduce the physical and chemical environment into an optical signal. Sensing voltage is our first target. Neuroscientists have long dreamed of a genetically encoded sensor that gives an optical signal in response to a change in membrane potential, with the goal of imaging electrical activity of neurons in vivo. Such a molecule could also be used to probe membrane potentials in mitochondria, cardiac cells, or in other non-neuronal cells. Our strategy is completely different from previous approaches to optical voltage sensing, and has already shown promising results. The technical implementation involves a) protein engineering and directed evolution to optimize an electrochromic response, and b) design and construction of an ultrasensitive laser imaging system capable of detecting this response in living cells. The methodology developed for sensing pH and voltage will later be applied to other sensing modalities, such as chloride and membrane tension. Public Health Relevance: We are working to develop a new class of molecules that allow us to see changes in voltage or pH inside of single cells. Neurons use voltage to communicate, so the ability to see neuronal activity will provide insights into brain function.